Analysis of mtDNA, miR-155 and BACH1 expression in hearts from donors with- and without- Down syndrome

Abstract

Cancer patients with Down syndrome (DS) are at increased risk for anthracycline-related cardiotoxicity. Mitochondrial DNA (mtDNA) alterations in hearts with- DS may contribute to anthracycline-related cardiotoxicity. Cardiac mtDNA and the mtDNA⁴⁹⁷⁷ deletion were quantitated in samples with- (n = 11) and without- DS (n = 31). Samples with- DS showed 30% lower mtDNA (DS_{MT-ND1/18S ratio}: 1.48 ± 0.72 vs. non-DS_{MT-ND1/18S ratio}: 2.10± 1.59; p = 0.647) and 30% higher frequency of the _mtDNA⁴⁹⁷⁷ _deletion (DS% _{frequency mtDNA}⁴⁹⁷⁷ _deletion: 0.0086 ± 0.0166 vs. non-DS% _{frequency mtDNA}⁴⁹⁷⁷ _deletion: 0.0066 ± 0.0124, p= 0.514) than samples without- DS. The BACH1 and microRNA-155 (miR-155) genes are located in chromosome 21, and their products have demonstrated roles during oxidative stress. BACH1 and miR-155 expression did not differ in hearts with- and without- DS. An association between BACH1 and miR-155 expression was detected in hearts without- DS, suggesting alterations between BACH1-miR-155 interactions in the DS settings.

INTRODUCTION

Children with Down syndrome (DS) represent 15% of pediatric acute myeloid leukemia (AML) cases (Taub and Ravindranath, 2011). Patients with- DS and AML are treated with combination chemotherapy that typically includes anthracyclines (e.g., daunorubicin, idarubicin, and doxorubicin), cytarabine, and etoposide. Epidemiological studies have identified pediatric cancer patients with- DS as a population at particularly greater risk for anthracycline-related cardiotoxicity (Krischer et al., 1997, Grenier and Lipshultz, 1998, Ravindranath et al., 2005). For example, a report from the Children’s Oncology group has documented clinically symptomatic cardiomyopathy in 17.5% of DS-AML patients treated with anthracyclines (O’Brien et al., 2008). Congenital heart disease occurs in 40–60% of patients with- DS and could increase the risk of developing anthracycline-related cardiotoxicity. However, development of anthracycline-related cardiotoxicity has also been observed in approximately 50% of children with- DS and AML without a diagnosis of congenital heart disease (O’Brien et al., 2008). A safe dose of anthracyclines for pediatric cancer patients with- DS remains to be defined, and the factors that determine the increased risk for anthracycline-related cardiotoxicity in children with- DS are unknown.

A cumulus of research has shown that cardiac mitochondria are key targets during the pathogenesis of anthracycline-related cardiotoxicity. For example, anthracyclines cause an irreversible decrease in mitochondrial Ca²⁺ loading and ATP content as well as impairment of membrane binding, assembly, and activity of mitochondrial creatine kinase. Anthracyclines can also lead to increased reactive oxygen species generation in cardiac mitochondrion, which can cause mitochondrial dysfunction [reviewed in: (Broder et al., 2008, Menna et al., 2008, Minotti et al., 2004)]. Several lines of evidence indicate that individuals with- DS have mitochondrial dysfunction (Coskun and Busciglio, 2012). Some of the major perturbations observed in mitochondria from DS cells and tissues include: 1) reduced redox activity and membrane potential, 2) ATP depletion, 3) changes in expression of genes involved in the Krebs cycle and oxidative phosphorylation, and 4) reduced activity of mitochondrial enzymes (Ogawa et al., 2002, Roat et al., 2007, Arbuzova et al., 2002, Infantino et al., 2011, Infantino et al., 2012, Pallardo et al., 2010). Evidence of mitochondrial dysfunction in the brains of patients with- DS and dementia (DSD) has been reported by Coskun et al. (2010). The authors documented decreased mitochondrial DNA (mtDNA) copy numbers in DSD brain tissue compared to brain tissue from age-matched controls (Coskun et al., 2010). Thus, it is reasonable to hypothesize that preexistent quantitative and/or qualitative alterations in cardiac mtDNA may contribute to the cardiotoxicity of anthracycline drugs in cancer patients with- DS. Conti et al. (2007) documented altered expression of mitochondrial genes in heart tissue from fetuses with trisomy 21 using oligonucleotide microarrays (Conti et al., 2007). To the best of our knowledge, the extent of quantitative and qualitative alterations in cardiac mtDNA in individuals with- DS has not been documented. Thus, the first aim of this study was to document mtDNA content in 11 heart samples from donors with- DS and 31 samples from donors without- DS. On the other hand, the presence of relatively high frequencies of the “common” mtDNA⁴⁹⁷⁷ deletion in various tissues has been associated with a number of distinct clinical phenotypes (e.g., Kearns-Sayre syndrome) and with the aging process (Shenkar et al., 1996). The mtDNA⁴⁹⁷⁷ deletion removes all or part of the genes encoding four complex I subunits, one complex IV subunit, two complex V subunits, and five tRNA genes. There are studies describing the frequency of the mtDNA⁴⁹⁷⁷ deletion in tissues (e.g., skeletal muscle, brain and heart) in both normal and pathological conditions (Meissner et al., 2008, Krishnan et al., 2008). However, it is not known whether DS status impacts the frequency of the mtDNA⁴⁹⁷⁷ deletion in heart tissue from affected individuals. Thus, the second aim of this study was to document the frequency of the “common” mtDNA⁴⁹⁷⁷ deletion in cardiac samples from donors with- and without- DS, respectively.

The altered cardiac expression of specific genes located in chromosome 21 coupled to the oxidative stress invoked by anthracycline drugs may increase the risk for anthracycline-related cardiotoxicity in individuals with- DS. BACH1 (Hsa21 band q22.11) and miR-155 (Hsa21 band q21.3) are of interest because of their roles during cellular oxidative stress. Briefly, the transcription regulator protein BACH1 binds to the promoter of genes containing antioxidant response elements (ARE) to repress cellular antioxidant responses which are mostly mediated by the master transcription factor Nrf2 (nuclear factor [erythroid-derived 2]-like 2, official symbol: NFE2L2) (Sykiotis and Bohmann, 2010). miR-155 has distinct tissue expression profiles and is involved in physiological and pathological processes (e.g., hematopoietic lineage differentiation, inflammation, cancer, and cardiovascular diseases) through interactions with various cellular targets (Faraoni et al., 2009). Of note, miR-155 binds to the 3′ untranslated region (3′-UTR) of BACH1 mRNA to inhibit the translation of the BACH1 protein (Pulkkinen et al., 2011). To date, there is a paucity of reports documenting BACH1 mRNA and miR-155 expression levels in hearts from donors with- DS. Thus, the third aim of this study was to document the cardiac expression of BACH1 mRNA and miR-155 in samples from donors with- and without- DS by using quantitative real-time polymerase chain reaction (qRT-PCR).

METHODS

Heart Samples

The Institutional Review Board of the State University of New York at Buffalo approved this research. Heart samples from donors with- (n = 11) and without- DS (n = 31) were procured from The National Disease Research Interchange (NDRI, funded by the National Center for Research Resources), The Cooperative Human Tissue Network (CHTN, funded by the National Cancer Institute), and the National Institute of Child Health and Human Development (NICHD) Brain and Tissue Bank. The postmortem to tissue recovery interval was ≤ 10 h. Samples (2–20 g, myocardium, left ventricle only) were frozen immediately after recovery and stored in liquid nitrogen until further processing. DS status (yes/no) was obtained from anonymous records provided by NDRI, CHTN, and NICHD. High quality DNA and RNA was isolated with an automatic QuickGene-810 purification system equipped with the appropriate DNA/RNA kits (Fujifilm Life Sciences). Purity and integrity of the RNA templates was assessed by measuring A₂₆₀/A₂₈₀ ratio and by gel electrophoresis in denaturing conditions following MIQE guidelines (Bustin et al., 2009).

Array CGH Analysis

Briefly, genomic DNA (3.0 μg) from test heart samples and an euploid reference sample was fluorescently labeled and hybridized to high resolution Agilent 244K aCGH arrays containing +236,000 coding and non-coding human probes. Changes in DNA copy number were determined by evaluating log₂ ratios across whole chromosomes. aCGH assays were performed at the Genomics core facility, Roswell Park Cancer Institute (Buffalo, NY).

Analysis of mtDNA content

Cardiac mtDNA content was measured using qRT-PCR. The primers for amplification of the mitochondrial gene MT-ND1 and the nuclear gene 18S rRNA are listed in Table 2. Reaction mixtures contained SYBR Advantage qPCR Premix (Clontech, Mountain View, CA), forward and reverse primers for either MT-ND1 or 18S rRNA, and total DNA template (10 ng). The qRT-PCR conditions for the amplification of both genes analyzed were as follows: 95°C for 30 seconds, followed by 40 cycles at 95°C for 6 seconds and 60°C for 34 seconds. Each qRT-PCR run included appropriate negative controls and a 6-point calibration curve made with diluted diploid cardiac DNA (range: 0.001 – 100 ng). Samples and standards for calibration curves were analyzed in triplicates in an iQ5 thermal cycler (Bio-Rad, Berkeley, CA) and data were averaged for analysis. Cycle threshold values (Ct) for MT-ND1 and 18S rRNA were plotted versus DNA content to evaluate the calibration curves. Linear regression coefficient values (r²) were ≥ 0.99. For each sample, MT-ND1/18S rRNA RNA ratios were calculated based on values extrapolated from the calibration curves.

Table 2.

Demographics of heart donors

	Non-DS	DS
Number of subjects (age-matched group)	31 21	11 8
Mean age (years) (age-matched group)	64.5 57.3	39.0 52.1
Standard deviation of age (age-matched group)	16.4 14.4	26.6 16.8
Males (age-matched group)	14 10	10 8
Females (age-matched group)	17 11	1 0
Subjects with myocardial infarction (age-matched group)	3 2	0 0
Subjects with heart failure (age-matched group)	2 2	0 0

Analysis of mtDNA⁴⁹⁷⁷ deletion

The frequency of the mtDNA⁴⁹⁷⁷ deletion in heart was determined by qRT-PCR with specific primers. Primer set “I” was developed to produce a 121 bp amplicon from a highly conserved region in the mitochondrial genome, while primer set “D” was designed to amplify a 169 bp amplicon from mtDNA containing the mtDNA⁴⁹⁷⁷ deletion (Table 2). The qRT-PCR amplification conditions for both amplicons were as follow: 95°C for 3 minutes, followed by 40 cycles at 95°C for 15 seconds, 64°C for 30 seconds and 72°C for 30 seconds. During the PCR cycle, the short extension time of 30 seconds favored amplification of the 169 bp amplicon from deleted mtDNA. PCR products were cloned and quantified to produce copy number standards for calibration curves. Each qRT-PCR run included appropriate negative controls and a 6-point calibration curve (range: 3.00 X 10² – 3.00 X 10⁷ copies). Samples and standards for calibration curves were analyzed in triplicates and data were averaged for analysis. For each sample, averaged Ct values from the “D” and “I” amplicons were used to extrapolate mtDNA copy numbers from the calibration curves (r² > 0.99). The percent frequency of the mtDNA⁴⁹⁷⁷ deletion was obtained with Equation 1.

$$\%\text{frequency}4977\text{bp}\text{mtDNA}\text{deletion}=\frac{\text{Copy}\text{number}4977\text{bp}\text{mtDNA}\text{deletion}}{\text{Copy}\text{number}\text{total}\text{mtDNA}}\times 100$$ Equation 1

Quantification of miR-155 expression

Total cardiac RNA (100 ng) was reversed-transcribed using TaqMan MicroRNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA) following the manufacturer’s protocol. Four microliters of the diluted (1:50) reverse transcription mix were used as template for the quantification of miR-155 by qRT-PCR with TaqMan MicroRNA Assays (Applied Biosystems). qRT-PCR reaction mixtures were incubated in an iQ5 thermal cycler (Bio-Rad). miR-155 (target) and U47 (reference) were amplified in parallel with the following cycling parameters 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. Calibration curves with diluted total RNA were prepared to analyze linearity (r²>0.97) and PCR efficiency (92% and 95%) for the amplification of miR-155 and U47, respectively. Samples and standards for calibration curves were analyzed in triplicates and data were averaged for analysis. For each sample, the averaged Ct values for miR-155 were normalized against the averaged Ct values for U47 using the dCt method (Schmittgen and Livak, 2008). The expression of miR-155 in individual heart samples was expressed relative to the average expression of miR-155 in heart samples from donors without- DS (n = 31), which was assigned an arbitrary value of 1.0.

Quantification of BACH1 mRNA expression

Cardiac BACH1 mRNA expression was analyzed by qRT-PCR with specific primers (Table 2). Briefly, total cardiac RNA (0.625 ng) was reverse transcribed and amplified with one-step QuantiTect SYBR Green RT-PCR kits (Qiagen, Venlo, The Netherlands). BACH1 and ACTB (reference gene) were amplified in parallel with the following cycling parameters: 50°C for 30 minutes (reverse transcription), 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, 56°C for 30 seconds and 72°C for 30 seconds. Calibration curves were prepared to analyze linearity (r² > 0.97) and PCR efficiency (94% and 97%) for the amplification of BACH1 and ACTB, respectively. For each sample, the averaged Ct values for BACH1 were normalized against the averaged Ct values for ACTB using the dCt method (Schmittgen and Livak, 2008). The expression of BACH1 in individual heart samples was expressed relative to the average expression of BACH1 in heart samples from donors without- DS (n = 31), which was assigned an arbitrary value of 1.0.

Data analysis

Statistics were computed with Excel 2007 (Microsoft Office; Microsoft, Redmond, WA) and GraphPad Prism version 4.03 (GraphPad Software Inc., La Jolla, CA). The Kolmogorov–Smirnov test was used to analyze the normality of datasets. The Mann-Whitney U test was used to compare group means. Pearson’s product-moment correlation coefficient was used for the analysis of normally distributed data and Spearman’s rank correlation coefficient was used for the analysis of non-normally distributed data. Data are expressed as the mean ± standard deviation (SD). Differences between means were considered to be significant at p < 0.05.

RESULTS

The main demographics from donors with- and without- DS are summarized in Table 1. In all cases, DS status (i.e., trisomy 21) was confirmed by aCGH. First, cardiac mtDNA content was examined by measuring MT-ND1/18S ratios in samples from donors with- and without- DS. On average, cardiac mtDNA content was 30% lower in samples from donors with- DS in comparison to samples from donors without- DS (DS_{MT-ND1/18S ratio}: 1.48 ± 0.72 vs. non-DS_{MT-ND1/18S ratio}: 2.10 ± 1.59; Mann-Whitney test, p = 0.647. Figure 1). Two age-matched groups were defined for the purpose of exploring possible age-related variability in the data (Table 1). Comparisons between the age-matched groups showed that the average cardiac mtDNA content was 33% lower in samples from donors with- DS than in samples from donors without- DS (DS_{MT-ND1/18S ratio}: 1.38 ± 0.45 vs. non-DS_{MT-ND1/18S ratio}: 2.06 ± 1.68; Mann-Whitney test, p = 0.902. Figure 1); however, the difference between means did not reach statistical significance at p < 0.05. Further correlation analyses showed no association between cardiac mtDNA content and age in donors with- (Pearson’s correlation coefficient, r_P = −0.298, p = 0.374) and without- DS (Spearman’s correlation coefficient, r_s = −0.222, p = 0.230. Figure 1).

Table 1.

List of primers used for PCR amplification.

MITOCHONDRIAL CONTENT
MT-ND1 forward	5′-CCCTAAAACCCGCCACATCT-3′
MT-ND1 reverse	5′-GAGCGATGGTGAGAGCTAAGGT-3′
18s rRNA forward	5′-TCAAGAACGAAAGTCGGAGG -3′
18s rRNA reverse	5′-GGACATCTAAGGGCATCACA -3′
mtDNA4977DELETION
PRIMER I forward	5′-TACTACAACCCTTCGCTGAC-3′
PRIMER I reverse	5′-AGTAGAAGAGCGATGGTGAGAGC-3′
PRIMER D forward	5-′CACCTCTTTACAGTGAAATGC-3′
PRIMER D reverse	5′-GAGGAAAGGTATTCCTGCTAATGC-3′
BACH1 EXPRESSION
BACH1 forward	5′-CAAGAATGTGAGGTAAAACTGCC-3′
BACH1 reverse	5′-GCTCTCCTTTTCACTTTGCAGC-3′
ACTB forward	5′-GGACTTCGAGCAAGAGATGG-3′
ACTB reverse	5′-AGCACTGTGTTGGCGTACAG-3′

Figure 1.

(A) MT-ND1/18S ratios in heart samples from donors with- (n = 11) and without- DS (n = 31). Top insert: MT-ND1/18S ratios in age-matched samples from donors with- and without- DS. Each symbol depicts the average of individual samples. Samples were analyzed in triplicates. Horizontal lines indicate group means. (B) Linear regression analysis of age vs. MT-ND1/18S ratio in heart samples from donors with- and without- DS. Top insert: linear regression analysis of age vs. MT-ND1/18S ratio in age-matched samples.

Second, the frequencies of the mtDNA⁴⁹⁷⁷ deletion were compared in cardiac samples from donors with- and without- DS. The average frequency of the mtDNA⁴⁹⁷⁷ deletion was 30% higher in samples from donors with- DS than in samples from donors without- DS (Figure 2A); however, the difference was not statistically significant (DS_{% frequency mtDNA}⁴⁹⁷⁷_deletion: 0.0086 ± 0.0166 vs. non-DS_{% frequency mtDNA}⁴⁹⁷⁷_deletion: 0.0066 ± 0.0124; Mann-Whitney test, p= 0.514. Figure 2). Similar comparisons between the age-matched groups showed that the frequency of the mtDNA⁴⁹⁷⁷ deletion was 44% higher in samples from donors with- DS than in samples from donors without- DS (DS_{% frequency mtDNA}⁴⁹⁷⁷_deletion: 0.0107 ± 0.0181 vs. non-DS_{% frequency mtDNA}⁴⁹⁷⁷_deletion: 0.0074 ± 0.0149; Mann-Whitney test, p= 0.678. Figure 2). Correlation analysis showed a significant positive association between the frequency of the mtDNA⁴⁹⁷⁷ deletion and age in cardiac samples from donors with- DS (Spearman’s correlation coefficient, r_S = 0.806, p = 0.007. Figure 2B). Likewise, there was a trend towards a positive association between the frequency of the mtDNA⁴⁹⁷⁷ deletion and age in cardiac samples from donors without- DS (Spearman’s correlation coefficient, r_S = 0.326, p = 0.074. Figure 2).

Figure 2.

(A) Frequency of the mtDNA⁴⁹⁷⁷ deletion in heart samples from donors with- (n = 11) and without- DS (n = 31). Top insert: frequency of the mtDNA⁴⁹⁷⁷ deletion in age-matched samples from donors with- and without- DS. Each symbol depicts the average of individual samples. Samples were analyzed in triplicates. Horizontal lines indicate group means. (B) Linear regression analysis of age vs. frequency of the mtDNA⁴⁹⁷⁷ deletion in heart samples from donors with- and without- DS. Top insert: linear regression analysis of age vs. frequency of the mtDNA⁴⁹⁷⁷ in age-matched samples.

Third, the relative expression of BACH1 mRNA was compared between cardiac samples from donors with- and without- DS. The relative expression of BACH1 mRNA was similar between donors with- and without- DS (DS_{BACH1 mRNA}: 0.9 ± 0.8 relative fold vs. non-DS_{BACH1 mRNA}: 1.0 ± 1.4 relative fold; Mann-Whitney test, p = 0.483. Figure 3). Analysis of the age-matched groups revealed no significant differences in cardiac BACH1 mRNA content between donors with- and without- DS (p = 0.942). There were no significant associations between cardiac BACH1 mRNA expression and age in donors with- (Spearman’s regression coefficient, r_S = −0.073, p = 0.839) and without- DS (Spearman’s regression coefficient, r_S = −0.094, p = 0.614. Figure 3).

Figure 3.

(A) BACH1 mRNA expression in samples from donors with- (n = 11) and without- DS (n = 31). Each symbol depicts the average of individual samples. Samples were analyzed in triplicates. Horizontal lines indicate group means. (B) Linear regression analysis of age vs. BACH1 mRNA expression in heart samples from donors with- and without- DS.

Figure 4 shows the cardiac relative expression of miR-155 in samples from donors with- and without- DS. On average, the relative expression of miR-155 did not differ between donors with- and without- DS (DS_miR-155: 0.8 ± 0.5 relative fold vs. non-DS_miR-155: 1.0 ± 1.1 relative fold; Mann-Whitney test, p = 0.819. Figure 4).Also, cardiac miR-155 levels did not differ between the age-matched groups from donors with- and without- DS (p = 0.317). Further correlation analysis revealed no significant associations between cardiac miR-155 expression and age in donors with- (Spearman’s regression coefficient, r_S = −0.501, p = 0.122) and without- DS (Spearman’s regression coefficient, r_S = −0.077, p = 0.680. Figure 4). The study by Pulkkinen et al. pinpointed miR-155 as a regulator of BACH1 expression (Pulkkinen et al., 2011). Thus, linear regression analysis was used to explore any potential association between cardiac BACH1 mRNA and miR-155 expression in samples from donors with- and without- DS. There was no significant association between BACH1 mRNA and miR-155 expression in samples from donors with- DS (Spearman’s regression coefficient, r_S= −0.064, p = 0.860). In contrast, there was a significant association between BACH1 mRNA and miR-155 expression in samples from donors without- DS (Spearman’s regression coefficient, r_S = 0.535, p = 0.002. Figure 5).

Figure 4.

(A) miR-155 expression in samples from donors with- (n = 11) and without- DS (n = 31). Each symbol depicts the average of individual samples. Samples were analyzed in triplicates. Horizontal lines indicate group means. (B) Linear regression analysis of age vs. miR-155 expression in heart samples from donors with- and without- DS.

Figure 5.

Linear regression analysis of BACH1 mRNA vs. miR-155 expression in hearts from donors without- DS (n = 31).

DISCUSSION

We hypothesized that pre-existent quantitative and/or qualitative alterations in cardiac mtDNA may contribute to the increased risk of anthracycline-related cardiotoxicity documented for cancer patients with- DS (Krischer et al., 1997, Grenier and Lipshultz, 1998, Ravindranath et al., 2005, O’Brien et al., 2008). Our pilot observations suggest that cardiac mtDNA content in samples from donors with- DS tends to be lower than in samples from donors without- DS (e.g., ≈ 33% decrease in the subset of age-matched samples. Figure 1). The difference between means did not reach statistical significance at α = 0.05; however, the trend may reflect the presence of quantitative differences in cardiac mtDNA content by DS status of potential pathophysiological relevance. For example, Larsson et al. showed that reductions in mtDNA content by ≈ 37% in the hearts of mice with a heterozygous deletion for the mitochondrial transcription factor A (TFAM) decreased the activity of mitochondrial complex I enzymes by ≈ 40% (Larsson et al., 1998). The authors speculated that the resulting impairment in mitochondrial respiratory chain function may contribute to precipitating cardiac dysfunction in response to environmental insults or physiological stressors. Also, Hsu et al. proposed that mtDNA content plays an important role in modulating the response to anthracycline treatment (Hsu et al., 2010). In this context, it is reasonable to hypothesize that slightly reduced cardiac mtDNA content may contribute to the risk for anthracycline-related cardiotoxicity in cancer patients with- DS. On the other hand, mtDNA content can decrease with age in some tissues. For example, Coskun et al. reported declines in mtDNA content with aging in brain samples from donors with- and without- DS (Coskun and Busciglio, 2012). In contrast, Miller et al. documented no significant decreases in mtDNA content with aging in cardiac tissue from “normal” donors (age range: neonate - 90 years old) (Miller et al., 2003). We observed no significant declines in cardiac mtDNA content with aging in donors with- (age range: 1 – 72 years old) and without- DS (age range: 19 – 97 years old. Figure 1). However, the slopes of the regression lines suggest that the decline in cardiac mtDNA content with aging is more pronounced in donors without- DS than in donors with- DS (Figure 1). It is interesting to note that the trend towards decreased cardiac mtDNA content in donors with- DS seems to be independent from the donor’s age and encompasses a relatively wide range of age (Figure 1). We also observed a non-significant ≈ 44% increase in the frequency of the “common” mtDNA⁴⁹⁷⁷ deletion in cardiac samples from age-matched donors with- DS in comparison to the frequency in samples from donors without- DS (Figure 2). On average, this translates to 1 copy of “deleted” mtDNA per 9345 total mtDNA copies (DS) and 1 copy of “deleted” mtDNA per 13513 total mtDNA copies (non-DS), respectively. Consistent with previous reports, the frequency of the “common” 4977 bp mtDNA deletion in cardiac tissue from relatively young donors (i.e., <30 years old) was low (Figure 2) (Marin-Garcia et al., 1996, Mohamed et al., 2006). The slopes of the lines in figure 2 suggest that the mtDNA⁴⁹⁷⁷ deletion accumulates faster with aging in hearts from subjects with- DS than in hearts from donors without- DS (Figure 2). Also and based on the extent the linear associations (i.e., DS, r_S = 0.806, p = 0.007, and non-DS, r_S = 0.326, p = 0.074), there seems to be a relatively strong correlation between aging and accumulation of the mtDNA⁴⁹⁷⁷ deletion in cardiac tissue from subjects with- DS (Figure 2). The mtDNA⁴⁹⁷⁷ deletion has been related to maternally inherited myopathy and with heart failure after myocardial infarction (Ide et al., 2001, Shanske et al., 2002). Accumulation of the deletion has been also correlated with dysfunction in the respiratory chain (Lezza et al., 1994). A recent report showed that the anthracyclines doxorubicin and daunorubicin induce dysfunction in the respiratory chain due to specific alterations in the mitochondrial proteome (Sterba et al., 2011). Thus, the increased risk for anthracycline-related cardiotoxicity in subjects with- DS may be due in part to the combination of higher cardiac accumulation of the mtDNA⁴⁹⁷⁷ deletion and lower cardiac mtDNA content, when compared to subjects without- DS.

We have shown increased expression of the anthracycline metabolizing enzyme CBR1 in hearts from donors with- DS (Kalabus et al., 2010). The CBR1 gene is located in the so-called “DS critical region” of chromosome 21 (21q22.12), and its product is involved in the intracardiac synthesis of cardiotoxic anthracycline alcohol metabolites (Menna et al., 2007). The altered expression of other chromosome 21 genes due to gene dosage effect may contribute to precipitate anthracycline-related cardiotoxicity in subjects with- DS. Thus, our second goal was to examine whether the cardiac expression of BACH1 and miR-155 showed increased expression in heart samples from donors with- DS due to gene dosage effects that could lead in consequence to an imbalance in the response to the oxidative damage induced by the treatment with anthracyclines (Kluza et al., 2004, Pereira et al., 2011). Our data suggest that the expression levels of BACH1 and miRNA-155 are not increased in hearts from donors with- DS (Figures 3 and 4). In this regard, it appears that natural interindividual variation in the cardiac expression of BACH1 (range: 0.2–3.1 relative fold, coefficient of variation, CV: 0.83) and miR-155 (range: 0.2–1.7 relative fold, coefficient of variation CV: 0.63) in samples from donors with- DS modulates the expected 1.5 fold increase due to trisomy 21. This finding is supported by the comprehensive study by Prandini et al. assessing gene-expression variation in lymphoblastoid cell lines (n = 14) and fibroblasts (n = 17) with- and without- trisomy 21 (Prandini et al., 2007). The authors found that only 39% and 62% of genes in lymphoblastoid and fibroblast cells, respectively, showed statistically significant overexpression in samples with trisomy 21 compared to euploid samples. Interestingly, BACH1 showed increased expression in trisomic fibroblasts (1.6 relative fold), but not in trisomic lymphoblasts (1.2 relative fold). Thus, our findings further emphasize the importance of considering interindividual variation in gene expression when evaluating the potential impact of a particular gene product in a trisomic context. Besides, we identified a positive association between BACH1 mRNA and miR-155 expression in heart samples from donors without- DS (Figure 5). Recently, Pulkkinen et al. reported inhibition of BACH1 protein translation by miR-155 in endothelial cells, which in turn resulted in protection against oxidative stress (Pulkkinen et al., 2011). Thus, the positive association in expression levels may reflect a plausible interaction between BACH1 and miR-155 in cardiac tissue from donors without- DS. In the myocardium of euploid subjects, increasing levels of the inhibitor miRNA-155 would be necessary to target increasing levels of the BACH-1 mRNA transcript. In contrast, the lack of a significant association between BACH1 mRNA and miR-155 levels in samples from donors with- DS may be indicative of a disruption in this layer of control against oxidative stress in the DS setting.

There are limitations in this pilot study, the main one being the small number of cardiac samples from donors with- DS. The scarcity of DS heart samples makes subgroup analysis and large-scale quantitative and statistical comparisons difficult. Our procurement rates for samples from donors with- DS is low (≈ 1 sample every 9 – 12 months), even after working with national cooperative resources such as NDRI, CHTN, and the NICHD Brain and Tissue Bank. This limitation is not exclusive to our efforts. A recent report from the Down Syndrome National Conference on Patient Registries, Research Databases, and Biobanks highlighted the fact that sample size limitations continue to impair research on vital issues affecting individuals with- DS (Oster-Granite et al., 2011, McCabe and McCabe, 2011). Second, we have not examined the presence of cardiac mtDNA sequence variants with pathogenic potential. Determining the amount of heteroplasmy (i.e., the percentage of mutant mtDNA relative to “wild type” mtDNA) is of paramount importance for inferring genotype-phenotype correlations. Developments in massively parallel DNA sequencing allow the detection of mutations in mtDNA (i.e., heteroplasmy) at the ≥5% level (Tang and Huang, 2010). Thus, future research should analyze the presence of mutations in cardiac mtDNA from donors with- and without- DS. Third, determinations of the effect size of potential co-variables (e.g., medication use, smoking status, concomitant diseases) were hindered by the combined effect of sample size limitations and incomplete clinical records from some donors. For example, it has been shown that cardiac ischemia could have an impact on mtDNA⁴⁹⁷⁷ deletion frequency (Wallace, 1992). However, we were unable to examine the potential impact of cardiac ischemia on mtDNA⁴⁹⁷⁷ deletion frequency because the level of detail included in the heart samples’ clinical background information was insufficient to conduct a separate analysis. Nevertheless, we believe that the collection of samples that is the subject of this study represents an informative window to explore potential tissue specific determinants for anthracycline-related cardiotoxicity in subjects with- DS. We propose that future research should further examine whether: a) decreased cardiac mtDNA content and increased mtDNA⁴⁹⁷⁷ deletion frequency, and b) BACH1-miRNA-155 interactions contribute to the increased risk of anthracycline-related cardiotoxicity in individuals with- DS.